Project Summary/Abstract Heparan sulfate (HS) represents a large class of polysaccharides that are expressed by nearly all mammalian cells. HS is comprised of a repeating disaccharide backbone that is subject to a range of modifications making this class of macromolecules one of the most information dense in all of biology. HS binds and regulates the function of many proteins including growth factors, cytokines, proteolytic enzymes and extracellular matrix proteins; however the underlying structural basis for this range of actions is not well understood. We hypothesize that the function of HS emerges from the arrangement and density of particular domain patterns (i.e, a local arrangement of various sequence clusters) and is not simply based on the existence of unique protein binding sites. To test this hypothesis we have developed the foundation of a computational method (ChainMaker Program (ChamP)) for deciphering the domain pattern of complex mixtures of HS chains based on disaccharide compositional analysis after heparinase digestion, expanded the use of molecular dynamics to explore the interaction of long HS chains with proteins of interest, and have developed a robust surface plasmon resonance method to evaluate HS-protein binding. Thus, the goal of this project is to use these tools to uncover the structural requirements for heparan sulfate (HS)-protein binding. To achieve this goal the digestion probabilities of heparin lyases will be refined using chemoenzymatically synthesized HS oligosaccharides of defined structure and the capabilities of ChamP will be expanded by including mammalian heparanase, nitrous acid digestion and additional analytic data. Using defined HS oligosaccharides and HS samples analyzed by ChamP in conjunction with molecular docking, molecular dynamics simulations, and surface plasmon resonance (SPR), the structural requirement for HS to bind and modulate a range of heparin-binding proteins will be determined. Aim 1 will expand, refine and validate ChamP for HS analysis using a set of defined HS oligosaccharides to train the program. ChamP will also be used to evaluate the structure of a set of tissue-derived HS samples. Aim 2 will evaluate the ability of HS oligosaccharides and HS preparations to bind to a range of proteins and will correlate activity with specific domain arrangements revealed by ChamP. In Aim 3 HS-protein binding will be investigated using molecular docking and molecular dynamics simulations to gain insight into how the arrangement of particular structural domains provides specificity. At the end of this project we will have a better understanding of the structural characteristics that define HS-protein control. This information can be used to identify active structures in HS that might be targeted or mimicked by new pharmaceuticals for disease treatment.